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Precision
Published in Lawrence S. Chan, William C. Tang, Engineering-Medicine, 2019
Spacer Acquisition. This step is critical in lining up Cas9 enzyme with the specific cutting sites. The CRISPR/Cas9 system contains a guide RNA (gRNA, also termed crRNA), which is a stretch of about 20 RNA bases and is complementary to the DNA target. In the natural bacteria defense, this gRNA will be complementary to the invading viral genome. In the medically indicated genome editing, this gRNA will be engineered to be complementary to the targeted DNA, be that a mutated gene in a genetic disease or a critical gene in a cancer cell. Situated at the end of a long RNA scaffold, gRNA locates and binds the target DNA, thus ensuring the correct DNA cutting site for Cas9 enzyme.
Health Professionals and Modern Human Research Ethics
Published in Howard Winet, Ethics for Bioengineering Scientists, 2021
The targeting ability of one of the Cas nucleases, Cas9 (from Streptococcus pyogenes) attached to a synthetic RNA complex (crRNA+tracrRNA), often referred to as a “guide RNA”, is crucial to the editing process. The ability of Cas9 to cleave almost any DNA site preceding a chemical signature called a PAM (Protospacer-Adjacent Motif), has made this nuclease a prime tool for the first step in genome editing (Salsman and Dellaire 2016). The CRISPR-Cas9 system has been used by a number of laboratories for mammalian cell genome editing since appearance of the “seminal” (Salsman and Dellaire 2016) publication of its success with DNA in 2012 (Jinek et al. 2012).
Genetic Engineering in Improving the Output of Algal Biorefinery
Published in Shashi Kant Bhatia, Sanjeet Mehariya, Obulisamy Parthiba Karthikeyan, Algal Biorefineries and the Circular Bioeconomy, 2022
Yogita Sharma, Ameesh Dev Singh, Sanjeet Mehariya, Obulisamy Parthiba Karthikeyan, Gajendra Pal Singh, Chandra Pal Singh, Antonio Molino
Despite their high accuracy, TALEN construction is both a time-consuming and an expensive process. Typically, to ensure specificity, at least 20 TAL effectors are designed for each target DNA, which differs only in their two amino acid residues (Nawaly et al., 2020). To overcome these problems Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR/Cas9) have been developed as a novel mechanism for introducing DSB in target DNAs in a much more simplified manner (Hsu et al., 2014). CRISPR are repetitive sequences found in bacteria and archaea that protect their genome from viral attacks, thus preventing DNA damage. CRISPR-associated Cas9 are endonuclease proteins that can form a ribonucleic protein (RNP) complex with a guide RNA (gRNA). This gRNA binds to a target DNA via the Watson and Crick model of nucleotide base pairing; meanwhile Cas9 induces site-specific DSBs (Barrangou and Oost, 2013). In microalgae, this technology has been used for enhancing the biorefinery productivity of desired compounds. Targeting certain genes via CRISPR/Cas9 has resulted in highly reproducible transformants of different algal species. C. reinhardtii has been the first algal model to be studied for gene knockout via CRISPR tool. The earliest study conducted on C. reinhardtii yielded poor results due to Cas9 toxicity in mutants (Jiang et al., 2014) following which several methods were devised to overcome this challenge. Most of the solutions were based on mutating the Cas-protein and direct delivery of RNP at the target site. Kao and Ng (2017) developed a proof-of-concept for CRISPRi-mediated knockdown of phosphoenol pyruvate carboxylase (PEPC) gene in C. reinhardtii. Mutants showed 94.2% increased lipid productivity compared to wild type (Kao and Ng, 2017). Similarly, omega-3 fatty acid desaturase was targeted via CRISPR/Cas9 in Chlorella vulgaris and a 46% w/w increase in lipid content was observed. This was the first time when CRISPR was used in Chlorella for genetic manipulation (Lin and Ng, 2020). Thalassiosira pseudonana, a marine diatom, has also been engineered successfully using a mutated version of Cas9, i.e., Cas9 nickase. A putative θ-type carbonic anhydrase gene was found to be successfully transformed (Nawaly et al., 2020). Other microalgae including Nannochloropsis, Synechocysti, Synechococcus, and Tetraselmis have also been edited via CRISPR-mediated mutagenesis (Chang et al., 2020; Naduthodi et al., 2019; Wendt et al., 2016; Yao et al., 2016). These findings clearly suggest that CRISPR/Cas9 is an efficient, hands-on, and precise tool for microalgal genetic engineering that can target specific genes in metabolic pathways to induce productivity, block competitive or intermediate metabolic steps, and can yield better results without altering the overall physiology of the cells.
The potential for the use of gene drives for pest control in New Zealand: a perspective
Published in Journal of the Royal Society of New Zealand, 2018
Peter K. Dearden, Neil J. Gemmell, Ocean R. Mercier, Philip J. Lester, Maxwell J. Scott, Richard D. Newcomb, Thomas R. Buckley, Jeanne M. E. Jacobs, Stephen G. Goldson, David R. Penman
The advent of CRISPR/Cas9 targeting technologies (Hsu et al. 2014) has given new life to the gene drive idea. CRISPR/Cas9 makes use of a prokaryotic system which allows cells to cut invasive DNA that has been encountered previously (Horvath & Barrangou 2010). The system consists of a nuclease, Cas9, that can be targeted to any sequence in the genome using a small RNA sequence called a guide RNA (gRNA), providing that target sequence sits next to a 2–6 base pair PAM motif (Horvath & Barrangou 2010). The combination of the Cas9 molecule, which cuts DNA to form double-stranded breaks, and specific gRNA that guide Cas9 to a particular sequence, provides the technology to cut DNA at specific locations (Beumer & Carroll 2014; Bassett & Liu 2014). Using gRNAs targeting a specific sequence in a pest genome, a gene drive mechanism using CRISPR/Cas9 would act in the same way as for HEGs (Esvelt et al. 2014).
Harnessing gene drive
Published in Journal of Responsible Innovation, 2018
John Min, Andrea L. Smidler, Devora Najjar, Kevin M. Esvelt
The basic mechanism of CRISPR genome editing involves supplying the CRISPR endonuclease, guide RNA(s) instructing it which sequences to cut, and templates encoding the edited sequences to be inserted. The cell then repairs the resulting double-strand break by incorporating the edited template sequence, a mechanism that is also utilized by naturally occurring homing nuclease gene drives. As a consequence, it is possible to build RNA-guided gene drive systems based on CRISPR endonucleases (Figure 2) (Esvelt et al. 2014).
Genome Modifying Reproductive Procedures and their Effects on Numerical Identity
Published in The New Bioethics, 2019
In this way, the guide RNA with a certain genetic code, searchers the specific genetic target of the DNA strand of the genome and then forms a complex with the Cas9 enzyme. This then cleaves the DNA strand and enables a specific genetic sequence to be taken out. The strand can then be rejoined or, alternatively, a new genetic sequence inserted.